This invention relates to polypeptide hormone analogues that exhibits enhanced pharmaceutical properties, such as increased thermodynamic stability, augmented resistance to thermal fibrillation above room temperature, decreased mitogenicity, and/or altered pharmacokinetic and pharmacodynamic properties, i.e., conferring more prolonged duration of action or more rapid duration of action relative to soluble formulations of the corresponding wild-type human hormone. More particularly, this invention relates to insulin analogues consisting of a single polypeptide chain that contains a novel class of foreshortened connecting (C) domains between A and B domains. Of length 6-11 residues, the C domains of this class consist of an N-terminal acidic element and a C-terminal segment derived from the connecting domain of human IGF-II. The single-chain insulin analogues of the present invention may optionally contain standard or non-standard amino-acid substitutions at other sites in the A or B domains.
The engineering of non-standard proteins, including therapeutic agents and vaccines, may have broad medical and societal benefits. Naturally occurring proteins—as encoded in the genomes of human beings, other mammals, vertebrate organisms, invertebrate organisms, or eukaryotic cells in general—often confer multiple biological activities. A benefit of derivative proteins would be to achieve selective activity, such as decreased binding to homologous cellular receptors associated with an unintended and unfavorable side effect, such as promotion of the growth of cancer cells. Yet another example of a societal benefit would be augmented resistance to degradation at or above room temperature, facilitating transport, distribution, and use. An example of a therapeutic protein is provided by insulin. Wild-type human insulin and insulin molecules encoded in the genomes of other mammals bind to insulin receptors is multiple organs and diverse types of cells, irrespective of the receptor isoform generated by alternative modes of RNA splicing or by alternative patterns of post-translational glycosylation. Wild-type insulin also binds with lower affinity to the homologous Type 1 insulin-like growth factor receptor (IGF-1R).
An example of a further medical benefit would be optimization of the stability of a protein toward unfolding or degradation. Such a societal benefit would be enhanced by the engineering of proteins more refractory than standard proteins with respect to degradation at or above room temperature for use in regions of the developing world where electricity and refrigeration are not consistently available. Analogues of insulin consisting of a single polypeptide chain and optionally containing non-standard amino-acid substitutions may exhibit superior properties with respect to resistance to thermal degradation or decreased mitogenicity. The challenge posed by its physical degradation is deepened by the pending epidemic of diabetes mellitus in Africa and Asia. Because fibrillation poses the major route of degradation above room temperature, the design of fibrillation-resistant formulations may enhance the safety and efficacy of insulin replacement therapy in such challenged regions.
Administration of insulin has long been established as a treatment for diabetes mellitus. A major goal of conventional insulin replacement therapy in patients with diabetes mellitus is tight control of the blood glucose concentration to prevent its excursion above or below the normal range characteristic of healthy human subjects. Excursions below the normal range are associated with immediate adrenergic or neuroglycopenic symptoms, which in severe episodes lead to convulsions, coma, and death. Excursions above the normal range are associated with increased long-term risk of microvascular disease, including retinapathy, blindness, and renal failure.
Insulin is a small globular protein that plays a central role in metabolism in vertebrates. Insulin contains two chains, an A chain, containing 21 residues, and a B chain containing 30 residues. The hormone is stored in the pancreatic β-cell as a Zn2+-stabilized hexamer, but functions as a Zn2+-free monomer in the bloodstream. Insulin is the product of a single-chain precursor, proinsulin, in which a connecting region (35 residues) links the C-terminal residue of B chain (residue B30) to the N-terminal residue of the A chain (FIG. 1A). A variety of evidence indicates that it consists of an insulin-like core and disordered connecting peptide (FIG. 1B). Formation of three specific disulfide bridges (A6-A11, A7-B7, and A20-B19; FIGS. 1A and 1B) is thought to be coupled to oxidative folding of proinsulin in the rough endoplasmic reticulum (ER). Proinsulin assembles to form soluble Zn2+-coordinated hexamers shortly after export from ER to the Golgi apparatus. Endoproteolytic digestion and conversion to insulin occurs in immature secretory granules followed by morphological condensation. Crystalline arrays of zinc insulin hexamers within mature storage granules have been visualized by electron microscopy (EM). The sequence of insulin is shown in schematic form in FIG. 1C. Individual residues are indicated by the identity of the amino acid (typically using a standard three-letter code), the chain and sequence position (typically as a superscript). Pertinent to the present invention is the invention of novel foreshortened C domains of length 6-11 residues in place of the 36-residue wild-type C domain characteristic of human proinsulin.
Fibrillation, which is a serious concern in the manufacture, storage and use of insulin and insulin analogues for the treatment of diabetes mellitus, is enhanced with higher temperature, lower pH, agitation, or the presence of urea, guanidine, ethanol co-solvent, or hydrophobic surfaces. Current US drug regulations demand that insulin be discarded if fibrillation occurs at a level of one percent or more. Because fibrillation is enhanced at higher temperatures, patients with diabetes mellitus optimally must keep insulin refrigerated prior to use. Fibrillation of insulin or an insulin analogue can be a particular concern for such patients utilizing an external insulin pump, in which small amounts of insulin or insulin analogue are injected into the patient's body at regular intervals. In such a usage, the insulin or insulin analogue is not kept refrigerated within the pump apparatus, and fibrillation of insulin can result in blockage of the catheter used to inject insulin or insulin analogue into the body, potentially resulting in unpredictable fluctuations in blood glucose levels or even dangerous hyperglycemia. Insulin exhibits an increase in degradation rate of 10-fold or more for each 10° C. increment in temperature above 25° C.; accordingly, guidelines call for storage at temperatures <30° C. and preferably with refrigeration. Fibrillation of basal insulin analogues formulated as soluble solutions at pH less than 5 (such as Lantus® (Sanofi-Aventis), which contains an unbuffered solution of insulin glargine and zinc ions at pH 4.0) also can limit their self lives due to physical degradation at or above room temperature; the acidic conditions employed in such formulations impairs insulin self-assembly and weakens the binding of zinc ions, reducing the extent to which the insulin analogues can be protected by sequestration within zinc-protein assemblies.
Insulin is susceptible to chemical degradation, involving the breakage of chemical bonds with loss of rearrangement of atoms within the molecule or the formation of chemical bonds between different insulin molecules. Such changes in chemical bonds are ordinarily mediated in the unfolded state of the protein, and so modifications of insulin that augment its thermodynamic stability also are likely to delay or prevent chemical degradation. Insulin is also susceptible to physical degradation. The present theory of protein fibrillation posits that the mechanism of fibrillation proceeds via a partially folded intermediate state, which in turn aggregates to form an amyloidogenic nucleus. In this theory, it is possible that amino-acid substitutions that stabilize the native state may or may not stabilize the partially folded intermediate state and may or may not increase (or decrease) the free-energy barrier between the native state and the intermediate state. Therefore, the current theory indicates that the tendency of a given amino-acid substitution in the two-chain insulin molecule to increase or decrease the risk of fibrillation is highly unpredictable. Models of the structure of the insulin molecule envisage near-complete unfolding of the three-alpha helices (as seen in the native state) with parallel arrangements of beta-sheets formed successive stacking of B-chains and successive stacking of A-chains; native disulfide pairing between chains and within the A-chain is retained. Such parallel cross-beta sheets require substantial separation between the N-terminus of the A-chain and C-terminus of the B-chain (>30 Å), termini ordinarily in close proximity in the native state of the insulin monomer (<10 Å). Marked resistance to fibrillation of single-chain insulin analogues with foreshortened C-domains is known in the art and thought to reflect a topological incompatibility between the splayed structure of parallel cross-beta sheets in an insulin protofilament and the structure of a single-chain insulin analogue with native disulfide pairing in which the foreshortened C-domain constrains the distance between the N-terminus of the A-chain and C-terminus of the B-chain to be unfavorable in a protofilament.
Single-chain insulin analogues might therefore seem to provide a favorable approach toward the design of fibrillation-resistant insulin analogues. However, in the past such analogues have exhibited low activities, which can be 1% or lower relative to wild-type human insulin. (Although Lee, H.C., et al. (2000) claimed that single-chain insulin analogues with wild-type A- and B-domains of length 57 residues or 58 residues exhibit receptor-binding affinities in the range 30-40% relative to human insulin, this publication was retracted in 2009 due to scientific misconduct; in our hands the analogues disclosed by Lee, H.C. et al. exhibit relative affinities of less than 1%.) Affinity might in part be restored by introduction of AspB10, a substitution known in the art to enhance the affinity of insulin for the insulin receptor. We have previously described a 57-residue single-chain insulin containing AspB10 with C-domain linker GGGPRR (SEQ ID NO: 20). However, use of foreshortened C-domains in conjunction with such substitutions in A-domain and/or B-domain can skew the ratio of binding toward an enhanced ratio of binding to IR-A relative to IR-B as disclosed in U.S. patent application Ser. No. 12/989,399, entitled “Isoform-Specific Insulin Analogues” (incorporated by reference herein). A single-chain insulin analogue with high receptor-binding affinity was described in which the foreshortened C-domain was the 12-residue C-domain of insulin-like growth factor I (IGF-I; sequence GYGSSSRRAPQT; SEQ ID NO: 12), yielding a chimeric protein. However, such chimeric molecules exhibit enhanced relative and absolute affinities for IGF-1R. Such alterations, like those associated with AspB10 and other substitutions at position B10, have elicited broad concern due to possible association with an increased risk of cancer in animals or human patients taking such analogues. This concern is especially marked with respect to basal insulin analogues, i.e., those designed for once-a-day administration with 12-24 hour profile of insulin absorption from a subcutaneous depot and 12-24 hour profile of insulin action.
The present invention was motivated by the medical and societal needs to engineer a basal once-a-day single-chain insulin analogue that combines (i) resistance to degradation with (ii) substantial in vivo hypoglycemic potency with (iii) reduced cross-binding to IGF-1R and (iv) a ratio of affinities for the A- and B isoforms of the insulin receptor that is similar to that of wild-type human insulin. The latter objective reflected the pleitropic functions and target tissues of insulin in the human body. The classical paradigm of insulin action has focused on organ-specific functions of adipocytes (where insulin regulates storage of fuels in the form of tryglyceride droplets), the liver (where insulin regulates the production of glucose via gluconeogenesis and regulates the storage of fuel in the form of glycogen) and muscle (where insulin regulates the influx of glucose from the bloodstream via trafficking to the plasma membrane of GLUT4) as the target tissues of the hormone. Recent research has revealed, however, that insulin has physiological roles in other organs and tissues, such as in the hypothalamus of the brain, wherein insulin-responsive neural circuitry influences hepatic metabolism, appetite, satiety, and possibly the set point for ideal body weight. Although the human genome contains a single gene encoding the insulin receptor, a transmembrane protein containing a cytoplasmic tyrosine-kinase domain, its pre-messenger RNA undergoes alterative splicing to yield distinct A and B isoforms, whose fractional distribution may differ from organ to organ and whose signaling functions may differ within the same cells.
The A and B isoforms (designated IR-A and IR-B) differ in affinity for insulin (affinity for IR-A is twofold higher than affinity for IR-B), and only IR-A (lacking a peptide domain in the alpha subunit encoded by exon 11) binds IGF-II with high affinity. Although insulin analogues are known in the art that differ from wild-type insulin in the ratio of respective affinities for IR-A and IR-B, it is possible that the safety and efficacy of insulin replacement therapy would optimally require administration of an insulin analogue whose ration of affinities for IR-A and IR-B is similar to that of wild-type insulin. Reduced binding of an insulin analogue to IR-A relative to IR-A, for example, might lead to a relative or absolute decrease in the extent of insulin signaling in the brain and in white blood cells, which express a predominance of IR-A receptors. Similarly, reduced binding an insulin analogue to IR-B relative to IR-B, for example, might lead to a relative or absolute decrease in the extent of insulin signaling in to classical target organs that exhibit predominance of IR-B receptors. Such skewed binding affinities might also perturb the cellular function of target cells (such as pancreatic beta-cells) in which IR-A-mediating insulin signaling and IR-B-mediated insulin signaling are thought to mediate different cellular functions that each contribute to proper beta-cell viability and secretory function. Because cancer cells can exhibit over-expression of IR-A, treatment of a patient with an analogue that exhibits enhanced potency of IR-A signaling (relative to wild-type human insulin) may pose a risk of increasing tumor growth. Mitogenic signaling by insulin analogues in cancer cells may be mediated by analogues that exhibit enhanced cross-binding to the mitogenic IGF-1R receptor (relative to wild-type human insulin) or by analogues that exhibit enhanced binding to IR-A (relative to wild-type human insulin) or by analogues that exhibit prolonged residence times on IGF-1R, IR-A, or IR-B (relative to wild-type human insulin). Basic residues near the C-terminus of the B-chain or B-domain (B28-B30), within a C-terminal extension (B31 or B32), or at the equivalent positions of a single-chain insulin analogue (C1 and C2) can enhance cross-binding of an insulin analogue to IGF-1R and thereby enhance mitogenicity.
It would be desirable, therefore, to invent single-chain insulin analogue with negligible mitogenicity and cross-binding to the IGF-1R that nonetheless retains at least a portion of the glucose-lowering effect of wild-type insulin. More generally, there is a need for an insulin analogue that displays increased thermodynamic stability and increased resistance to fibrillation above room temperature while exhibiting a ratio of affinities for the A- and B isoforms of the insulin receptor, and so by implication at least a subset of the multiple organ-specific biological activities of wild-type insulin.